Anti-factor b antibodies and their uses

ABSTRACT

The invention concerns the prevention and treatment of complement-associated eye conditions, such as choroidal neovascularization (CNV) and age-related macular degeneration (AMD), by administration of factor B antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/291,222 filed Nov. 6, 2008, which is a non-provisionalapplication filed under 37 CFR 1.53(b)(1), claiming priority under 35USC 119(e) to provisional application No. 61/002,648 filed Nov. 8, 2007,the contents of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention concerns anti-factor B antibodies and their use inthe prevention and treatment of complement-associated eye conditions,such as choroidal neovascularization (CNV), diabetic retinopathy andage-related macular degeneration (AMD).

BACKGROUND OF THE INVENTION

The complement system is a complex enzyme cascade made up of a series ofserum glycoproteins, that normally exist in inactive, pro-enzyme form.Two main pathways, the classical and the alternative pathway, canactivate complement, which merge at the level of C3, where two similarC3 convertases cleave C3 into C3a and C3b.

Macrophages are specialist cells that have developed an innate capacityto recognize subtle differences in the structure of cell-surfaceexpressed identification tags, so called molecular patterns (Taylor, etal., Eur J Immunol 33, 2090-2097 (2003); Taylor, et al., Annu RevImmunol 23, 901-944 (2005)). While the direct recognition of thesesurface structures is a fundamental aspect of innate immunity,opsonization allows generic macrophage receptors to mediate engulfment,increasing the efficiency and diversifying recognition repertoire of thephagocyte (Stuart and Ezekowitz, Immunity 22, 539-550 (2005)). Theprocess of phagocytosis involves multiple ligand-receptor interactions,and it is now clear that various opsonins, including immunoglobulins,collectins, and complement components, guide the cellular activitiesrequired for pathogen internalization through interaction withmacrophage cell surface receptors (reviewed by Aderem and Underhill,Annu Rev Immunol 17, 593-623 (1999); Underhill and Ozinsky, Annu RevImmunol 20, 825-852 (2002)). While natural immunoglobulins encoded bygermline genes can recognize a wide variety of pathogens, the majorityof opsonizing IgG is generated through adaptive immunity, and thereforeefficient clearance through Fc receptors is not immediate (Carroll, NatImmunol 5, 981-986 (2004)). Complement, on the other hand, rapidlyrecognizes pathogen surface molecules and primes the particle for uptakeby complement receptors (Brown, Infect Agents Dis 1, 63-70 (1991)).

Complement consists of over 30 serum proteins that opsonize a widevariety of pathogens for recognition by complement receptors. Dependingon the initial trigger of the cascade, three pathways can bedistinguished (reviewed by (Walport, N Engl J Med 344, 1058-1066(2001)). All three share the common step of activating the centralcomponent C3, but they differ according to the nature of recognition andthe initial biochemical steps leading to C3 activation. The classicalpathway is activated by antibodies bound to the pathogen surface, whichin turn bind the C1q complement component, setting off a serine proteasecascade that ultimately cleaves C3 to its active form, C3b. The lectinpathway is activated after recognition of carbohydrate motifs by lectinproteins. To date, three members of this pathway have been identified:the mannose-binding lectins (MBL), the SIGN-R1 family of lectins and theficolins (Pyz et al., Ann Med 38, 242-251 (2006)) Both MBL and ficolinsare associated with serine proteases, which act like C1 in the classicalpathway, activating components C2 and C4 leading to the central C3 step.The alternative pathway contrasts with both the classical and lectinpathways in that it is activated due to direct reaction of the internalC3 ester with recognition motifs on the pathogen surface. Initial C3binding to an activating surface leads to rapid amplification of C3bdeposition through the action of the alternative pathway proteasesfactor B and factor B. Importantly, C3b deposited by either theclassical or the lectin pathway also can lead to amplification of C3bdeposition through the actions of Factors B and D. In all three pathwaysof complement activation, the pivotal step in opsonization is conversionof the component C3 to C3b. Cleavage of C3 by enzymes of the complementcascades exposes the thioester to nucleophilic attack, allowing covalentattachment of C3b onto antigen surfaces via the thioester domain. Thisis the initial step in complement opsonization. Subsequent proteolysisof the bound C3b produces iC3b, C3c and C3dg, fragments that arerecognized by different receptors (Ross and Medof, Adv Immunol 37,217-267 (1985)). This cleavage abolishes the ability of C3b to furtheramplify C3b deposition and activate the late components of thecomplement cascade, including the membrane attack complex, capable ofdirect membrane damage. However, macrophage phagocytic receptorsrecognize C3b and its fragments preferentially; due to the versatilityof the ester-bond formation, C3-mediated opsonization is central topathogen recognition (Holers et al., Immunol Today 13, 231-236 (1992)),and receptors for the various C3 degradation products therefore play animportant role in the host immune response. C3 itself is a complex andflexible protein consisting of 13 distinct domains. The core of themolecule is made up of 8 so-called macroglobulin (MG) domains, whichconstitute the tightly packed α and β chains of C3. Inserted into thisstructure are CUB (C1r/C1s, Uegf and Bone mophogenetic protein-1) andTED domains, the latter containing the thioester bond that allowscovalent association of C3b with pathogen surfaces. The remainingdomains contain C3a or act as linkers and spacers of the core domains.Comparison of C3b and C3c structures to C3 demonstrate that the moleculeundergoes major conformational rearrangements with each proteolysis,which exposes not only the TED, but additional new surfaces of themolecule that can interact with cellular receptors (Janssen and Gros,Mol Immunol 44, 3-10 (2007)).

Age-related Macular Degeneration (AMD) is the leading cause of blindnessin the elderly worldwide. AMD is characterized by a progressive loss ofcentral vision attributable to degenerative and neovascular changes inthe macula, a highly specialized region of the ocular retina responsiblefor fine visual acuity. Recent estimates indicate that 14 millionpersons are blind or severely visually impaired because of AMD. Thedisease has a tremendous impact on the physical and mental health of thegeriatric population and their families and is becoming a major publichealth burden.

New discoveries, however, are beginning to provide a clearer picture ofthe relevant cellular events, genetic factors, and biochemical processesassociated with early AMD.

Factor B is a tightly regulated, highly specific serine protease. In itsactivated form, it catalyzes the central amplification step ofcomplement activation to initiate inflammatory responses, cell lysis,phagocytosis and B-cell stimulation (Carroll et al., Nat. Immunol.5:981-986 (2004)). factor B is activated through an assembly process: itbinds surface-bound C3b, or its fluid-phase counterpart C3(H₂O), afterwhich it is cleaved by factor B into fragments Ba (residues 1-234) andBb (residues 235-739) (Fishelson et al., J. Immunol. 132:1430-1434(1984)). Fragment Ba dissociates from the complex, leaving behind thealternative pathway C3 convertase complex C3b-Bb, which cleaves C3 intoC3a and C3b. This protease complex is intrinsically instable. Oncedissociated from the complex, Bb cannot reassociate with C3b (Pangburnet al, Bochem. J. 235:723-730 (1986)).

The proenzyme factor B consists of three N-terminal complement controlprotein (CCP) domains, connected by a 45-residue linker to a VWA domainand a C-terminal serine protease (SP) domain, which carries thecatalytic center. Striking differences from other serine proteases areobserved in the active center of factor B (Xu et al., Immunol. Rev. 180:123-135 (2001)). The catalytic triad residues Asp102, His57 and Ser195(chymotrypsinogen numbering has been used for the serine proteasedomain), and the three residues forming the non-specific substratebinding site, Ser-Trp-Gly214-216, have typical conformations. However,the oxyanion hole displays an inactive conformation similar to thatobserved in certain zymogens. This is primarily due to an inwardorientation of the carbonyl oxygen of Arg192, the backbone of which,along with those of Cys191, Gly193 and Asp194, adapts a single turn310-helix conformation. The carbonyl oxygen of Arg192 forms an H bondwith the amide group of Ser195, thus reducing the positive charge thatcharacterizes active oxyanion holes. This inactive configuration of theoxyanion hole is not compatible with the high catalytic activityexpressed by the C3 convertase. Accordingly, a conformational changeleading to realignment of the structural elements of the oxyanion holemust be induced by the co-factor, the substrate, or both.

The level of factor B is relatively high in the plasma is ˜1665 nM, butlow in the eye ˜29 nM). Therefore, it forms an attractive target forantibody therapy. The present invention provides anti-factor Bantibodies for the prevention and treatment of AMD and othercomplement-associated eye conditions.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the identificationof an anti-factor B antibody producing hybridoma that was selected outof a screen of 400 different clones based on its low IC50 value inhemolytic assays and on its cross-reactivity with cynomolgus factor B.

In one aspect, the invention concerns an anti-factor B antibody bindingessentially to the same epitope as anti-factor B antibody 1F7.

In one embodiment, the antibody is an anti-factor B antibody comprisingthe light and/or heavy chain hypervariable region sequences ofanti-factor B antibody 1F7 (SEQ ID NOs: 1 and 2, respectively).

In another embodiment, the antibody is an anti-factor B antibodycomprising the light and/or heavy chain variable region sequences ofanti-factor B antibody 1F7 (SEQ ID NOs: 1 and 2, respectively).

In yet another embodiment, the anti-factor B antibody is antibody 1F7comprising a light chain sequence of SEQ ID NO: 1 and the heavy chainsequence of SEQ ID NO: 2.

In a further embodiment, the anti-factor B antibody is an antibodyfragment, which may, for example, be selected from the group consistingof Fab, Fab′, F(ab′)₂, scFv, (scFv)₂, dAb, complementarity determiningregion (CDR) fragments, linear antibodies, single-chain antibodymolecules, minibodies, diabodies, and multispecific antibodies formedfrom antibody fragments.

In all embodiments, the antibody preferably is a monoclonal antibodywhich may, for example, be chimeric, humanized or human.

In another aspect, the present invention concerns a method for theprevention or treatment of a complement-associated eye conditioncomprising administering to a subject in need an effective amount of afactor B antagonist.

In various embodiments, the subject in need is a mammal, such as ahuman, and the factor B antagonist is selected from the group consistingof anti-factor B antibodies and fragments thereof, binding polypeptides,peptides, and non-peptide small molecules.

In a preferred embodiment, the factor B antagonist is an antibody or anantibody fragment, as hereinabove defined.

Complement-associated eye conditions include, for example, age-relaredmacular degeneration (AMD), choroidal neovascularization (CNV), uveitis,diabetic and other ischemia-related retinopathies, diabetic macularedema, pathological myopia, von Hippel-Lindau disease, histoplasmosis ofthe eye, Central Retinal Vein Occlusion (CRVO), cornealneovascularization, dry eye and retinal neovascularization.

In another aspect, the invention concerns a kit comprising a factor Bantagonist and instructions for administering said antagonist to treat acomplement-associated eye condition.

In yet another aspect, the invention concerns the use of a factor Bantagonist in the preparation of a medicament for the treatment of acomplement-associated eye condition.

In a further aspect the invention concerns a factor B antagonist for usein the treatment of a complement-associated eye condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Patent and Trademark Office upon request and paymentof the necessary fees.

FIG. 1 Method for dissection of human donor eyes for measuring levels ofcomplement components by ELISA

FIG. 2 Donor tissues used in the studies.

FIG. 3. Levels of factor B in vitreous and Bruch's obtained from normaland AMD donor eyes. Factor B levels were measured by a factor B-specificELISA as described. B: total levels of factor B in the eye weredetermined by calculating the total contribution of factor B expressedin Bruch's membrane and the total amount of factor B found in vitreous.

FIG. 4 Characterization of anti-factor B antibody 1F7 in a hemolyticassay selective for the alternative pathway of complement. IC50 and IC90values are indicated below and the assay was performed as described inthe methods section.

FIG. 5 Polypeptide sequences of the anti-factor B antibody 1F7 heavy andlight chains (SEQ ID NOS: 1 and 2), wherein the κ light chain constantregion sequence is shown in red and the heavy chain constant regionsequence is shown in green.

FIG. 6 Polypeptide sequence of human factor B polypeptide (SEQ ID NO:3).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

The terms “factor B” and “complement factor B” are used interchangeably,and refer to native sequence and variant factor B polypeptides. Othernames known in the art for complement factor B include B-factor,properdin; C3 proaccelerator; C3 proactivator; C3/C5 convertase; andglycine-rich beta-glycoprotein.

A “native sequence” factor B, is a polypeptide having the same aminoacid sequence as a factor B polypeptide derived from nature, regardlessof its mode of preparation. Thus, native sequence factor B can beisolated from nature or can be produced by recombinant and/or syntheticmeans. In addition to a mature factor B protein, such as a mature humanfactor B protein (SEQ ID NO: 3), the term “native sequence factor B”,specifically encompasses naturally-occurring precursor forms of factor B(e.g., an inactive preprotein, which is proteolytically cleaved toproduce the active form), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants offactor B, as well as structural conformational variants of factor Bmolecules having the same amino acid sequence as a factor B polypeptidederived from nature. Factor B polypeptides of non-human animals,including higher primates and non-human mammals, are specificallyincluded within this definition.

“Factor B variant” or “complement factor B variant” means an activefactor B polypeptide as defined below having at least about 80% aminoacid sequence identity: to a native sequence factor B polypeptide, suchas the native sequence human factor B polypeptide of SEQ ID NO: 3.Ordinarily, a factor B variant will have at least about 80% amino acidsequence identity, or at least about 85% amino acid sequence identity,or at least about 90% amino acid sequence identity, or at least about95% amino acid sequence identity, or at least about 98% amino acidsequence identity, or at least about 99% amino acid sequence identitywith the mature human amino acid sequence of SEQ ID NO: 3. Preferably,the highest degree of sequence identity occurs within the active site offactor B.

The crystal structure of the serine protease domain of factor B waspublished by Jing et al., EMBO J. 19:164-173 (2000); and the crystalstructure of factor B has been reported by Milder et al., Natl. Struct.Mol. Biol. 14(3):224-8 (2007). The “active site” of factor B is definedby the catalytic triad residues (H57, D102 and S195, using chymotrypsinnumbering) and the non-specific substrate-binding site (W215-G216).

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in a reference factor B sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference factor B-encoding sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes nucleic acid molecules contained in cells that ordinarilyexpress an encoded polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

An “isolated” factor B polypeptide-encoding nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the factor B-encoding nucleic acid. An isolatedfactor B polypeptide-encoding nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated factor Bpolypeptide-encoding nucleic acid molecules therefore are distinguishedfrom the encoding nucleic acid molecule(s) as they exists in naturalcells. However, an isolated factor B-encoding nucleic acid moleculeincludes factor B-encoding nucleic acid molecules contained in cellsthat ordinarily express factor B where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “antagonist” is used in the broadest sense, and includes anymolecule that is capable of neutralizing, blocking, partially or fullyinhibiting, abrogating, reducing or interfering with a factor Bbiological activity. Factor B antagonists include, without limitation,anti-factor B antibodies and antigen-binding fragments thereof, otherbinding polypeptides, peptides, and non-peptide small molecules, thatbind to factor B and are capable of neutralizing, blocking, partially orfully inhibiting, abrogating, reducing or interfering with factor Bactivities, such as the ability of factor B to participate in thepathology of a complement-associated eye condition.

A “small molecule” is defined herein to have a molecular weight belowabout 600, preferably below about 1000 daltons.

“Active” or “activity” or “biological activity” in the context of afactor B antagonist of the present invention is the ability theantagonize (patially or fully inhibit) a biological activity of factorB. A preferred biological activity of a factor B antagonist is theability to achieve a measurable improvement in the state, e.g.pathology, of a factor B-associated disease or condition, such as, forexample, a complement-associated eye condition. The activity can bedetermined in in vitro or in vivo tests, including binding assays, usinga relevant animal model, or human clinical trials.

The term “complement-associated eye condition” is used in the broadestsense and includes all eye conditions the pathology of which involvescomplement, including the classical and the alternative pathways, and inparticular the alternative pathway of complement. Complement-associatedeye conditions include, without limitation, macular degenerativediseases, such as all stages of age-related macular degeneration (AMD),including dry and wet (non-exudative and exudative) forms, choroidalneovascularization (CNV), uveitis, diabetic and other ischemia-relatedretinopathies, and other intraocular neovascular diseases, such asdiabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO),corneal neovascularization, and retinal neovascularization. A preferredgroup of complement-associated eye conditions includes age-relatedmacular degeneration (AMD), including non-exudative (wet) and exudative(dry or atrophic) AMD, choroidal neovascularization (CNV), diabeticretinopathy (DR), and endophthalmitis.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In treatment of an immune related disease,a therapeutic agent may directly alter the magnitude of response of acomponent of the immune response, or render the disease more susceptibleto treatment by other therapeutic agents, e.g., antibiotics,antifungals, anti-inflammatory agents, chemotherapeutics, etc.

The “pathology” of a disease, such as a complement-associated eyecondition, includes all phenomena that compromise the well-being of thepatient. This includes, without limitation, abnormal or uncontrollablecell growth (neutrophilic, eosinophilic, monocytic, lymphocytic cells),antibody production, auto-antibody production, complement production,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of any inflammatory or immunological response,infiltration of inflammatory cells (neutrophilic, eosinophilic,monocytic, lymphocytic) into cellular spaces, etc.

The term “mammal” as used herein refers to any animal classified as amammal, including, without limitation, humans, higher primates, domesticand farm animals, and zoo, sports or pet animals such horses, pigs,cattle, dogs, cats and ferrets, etc. In a preferred embodiment of theinvention, the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Therapeutically effective amount” is the amount of a “factor Bantagonist” which is required to achieve a measurable improvement in thestate, e.g. pathology, of the target disease or condition, such as, forexample, a complement-associated eye condition.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42 C; or (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide of the invention fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

The term “antibody” is used in the broadest sense and specificallycovers, without limitation, single anti-factor B monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies) andanti-factor B antibody compositions with polyepitopic specificity. Theterm “monoclonal antibody” as used herein refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J Mol.Biol. 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen (i.e.has a binding affinity (K_(d)) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁷ M and most preferably no more thanabout 1×10⁻⁷ M) but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but preferably is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Ina preferred embodiment, the antibody mutant will have an amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the species-dependent antibody, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). V_(H) refers to the variabledomain of the heavy chain. V_(L) refers to the variable domain of thelight chain. According to the methods used in this invention, the aminoacid positions assigned to CDRs and FRs may be defined according toKabat (Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991)). Amino acidnumbering of antibodies or antigen binding fragments is also accordingto that of Kabat.

As used herein, the term “Complementarity Determining Regions (CDRs;ie., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk (1987) J Mol. Biol. 196:901-917). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. For example, the CDRH1 of the heavy chain of antibody 4D5 includesamino acids 26 to 35.

“Framework regions” (hereinafter FR) are those variable domain residuesother than the CDR residues. Each variable domain typically has four FRsidentified as FR1, FR2, FR3 and FR4. If the CDRs are defined accordingto Kabat, the light chain FR residues are positioned at about residues1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and theheavy chain FR residues are positioned about at residues 1-30 (HCFR1),36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chainresidues. If the CDRs comprise amino acid residues from hypervariableloops, the light chain FR residues are positioned about at residues 1-25(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the lightchain and the heavy chain FR residues are positioned about at residues1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in theheavy chain residues. In some instances, when the CDR comprises aminoacids from both a CDR as defined by Kabat and those of a hypervariableloop, the FR residues will be adjusted accordingly. For example, whenCDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,eg. NNK, NNS, XYZ DVK and the like. A “non-random codon set”, as usedherein, thus refers to a codon set that encodes select amino acids thatfulfill partially, preferably completely, the criteria for amino acidselection as described herein. Synthesis of oligonucleotides withselected nucleotide “degeneracy” at certain positions is well known inthat art, for example the TRIM approach (Knappek et al. (1999) J Biol.296:57-86); Garrard & Henner (1993) Gene 128:103). Such sets ofoligonucleotides having certain codon sets can be synthesized usingcommercial nucleic acid synthesizers (available from, for example,Applied Biosystems, Foster City, Calif.), or can be obtainedcommercially (for example, from Life Technologies, Rockville, Md.).Therefore, a set of oligonucleotides synthesized having a particularcodon set will typically include a plurality of oligonucleotides withdifferent sequences, the differences established by the codon set withinthe overall sequence. Oligonucleotides, as used according to theinvention, have sequences that allow for hybridization to a variabledomain nucleic acid template and also can, but does not necessarily,include restriction enzyme sites useful for, for example, cloningpurposes.

The term “antibody fragment” is used herein in the broadest sense andincludes, without limitation, Fab, Fab′, F(ab′)₂, scFv, (scFv)₂, dAb,and complementarity determining region (CDR) fragments, linearantibodies, single-chain antibody molecules, minibodies, diabodies, andmultispecific antibodies formed from antibody fragments.

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)₂ antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman (1992) Curr. Opin.Struct. Biol. 3:355-362, and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells (1991) Methods: A companion to Methodsin Enzymology 3:205-0216.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, fl, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

As used herein, “solvent accessible position” refers to a position of anamino acid residue in the variable regions of the heavy and light chainsof a source antibody or antigen binding fragment that is determined,based on structure, ensemble of structures and/or modeled structure ofthe antibody or antigen binding fragment, as potentially available forsolvent access and/or contact with a molecule, such as anantibody-specific antigen. These positions are typically found in theCDRs and on the exterior of the protein. The solvent accessiblepositions of an antibody or antigen binding fragment, as defined herein,can be determined using any of a number of algorithms known in the art.Preferably, solvent accessible positions are determined usingcoordinates from a 3-dimensional model of an antibody, preferably usinga computer program such as the InsightII program (Accelrys, San Diego,Calif.). Solvent accessible positions can also be determined usingalgorithms known in the art (e.g., Lee and Richards (1971) J. Mol. Biol.55, 379 and Connolly (1983) J. Appl. Cryst. 16, 548). Determination ofsolvent accessible positions can be performed using software suitablefor protein modeling and 3-dimensional structural information obtainedfrom an antibody. Software that can be utilized for these purposesincludes SYBYL Biopolymer Module software (Tripos Associates). Generallyand preferably, where an algorithm (program) requires a user input sizeparameter, the “size” of a probe which is used in the calculation is setat about 1.4 Angstrom or smaller in radius. In addition, determinationof solvent accessible regions and area methods using software forpersonal computers has been described by Pacios (1994) Comput. Chem.18(4): 377-386.

DETAILED DESCRIPTION

Complement plays a crucial role in the body's defense, and, togetherwith other components of the immune system, protect the individual frompathogens invading the body. However, if not properly activated orcontrolled, complement can also cause injury to host tissues.Inappropriate activation of complement is involved in the pathogenesisof a variety of diseases, referred to as complement associated diseasesor disorders, such as immune complex and autoimmune diseases, andvarious inflammatory conditions, including complement-mediatedinflammatory tissue damage. The pathology of complement-associateddiseases varies, and might involve complement activation for a long orshort period of time, activation of the whole cascade, only one of thecascades (e.g. classical or alternative pathway), only some componentsof the cascade, etc. In some diseases complement biological activitiesof complement fragments result in tissue injury and disease.Accordingly, inhibitors of complement have high therapeutic potential.Selective inhibitors of the alternative pathway would be particularlyuseful, because clearance of pathogens and other organisms from theblood through the classical pathway will remain intact.

The factor B antagonists of the present invention are useful for theprevention and treatment of complement-associated eye conditions (alleye conditions and diseases the pathology of which involves complement,including the classical and the alternative pathways, and in particularthe alternative pathway of complement), such as, for example, maculardegenerative diseases, such as all stages of age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)forms, choroidal neovascularization (CNV), uveitis, diabetic and otherischemia-related retinopathies, endophthalmitis, and other intraocularneovascular diseases, such as diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, and retinalneovascularization. A preferred group of complement-associated eyeconditions includes age-related macular degeneration (AMD), includingnon-exudative (wet) and exudative (dry or atrophic) AMD, choroidalneovascularization (CNV), diabetic retinopathy (DR), andendophthalmitis.

AMD is age-related degeneration of the macula, which is the leadingcause of irreversible visual dysfunction in individuals over the age of60. Two types of AMD exist, non-exudative (dry) and exudative (wet) AMD.The dry, or nonexudative, form involves atrophic and hypertrophicchanges in the retinal pigment epithelium (RPE) underlying the centralretina (macula) as well as deposits (drusen) on the RPE. Patients withnonexudative AMD can progress to the wet, or exudative, form of AMD, inwhich abnormal blood vessels called choroidal neovascular membranes(CNVMs) develop under the retina, leak fluid and blood, and ultimatelycause a blinding disciform scar in and under the retina. NonexudativeAMD, which is usually a precursor of exudative AMD, is more common. Thepresentation of nonexudative AMD varies; hard drusen, soft drusen, RPEgeographic atrophy, and pigment clumping can be present. Complementcomponents are deposited on the RPE early in AMD and are majorconstituents of drusen.

The present invention specifically concerns the treatment of AMD,including category 3 and category 4 AMD. Category 3 AMD is characterizedby the absence of choroidal neovascularization in both eyes, at leastone eye having a visual acuity of 20/32 or better with at least onelarge druse (e.g. 125 μm), extensive (as measured by drusen area)intermediate drusen, or geographic atrophy (GA) that does not involvethe center of the macula, or any combination of these. Category 3 AMD(which is still considered “dry” AMD) has a high risk of conversion tochoroidal neovascularization (CNV) or geographic atrophy (GA).

Category 4 high risk dry AMD can be separated in two categories. 1. Thefellow eye has CNV. 2. The fellow eye does not have CNV. The 1stcategory is independent of any pathology and automatically defaults tohigh risk dry AMD if there is a fellow eye with CNV. In the 2^(nd)category pathology is characterized by large confluent drusen andmottling of the retinal pigmented epithelium (RPE). Category 4 high riskwet AMD is identified by the presence of CNV. In all category 4patients, visual acuity is 20/32 or worse Typically, high risk dry AMDof the 1^(st) category (fellow eye wet AMD), if untreated, rapidlyprogresses into choroidal neovascularization (CNV), at a rate about10-30-times higher than the rate of progression for category 3 (not highrisk) AMD.

Factor B antagonists find particular utility in the prevention of theprogression of AMD (in particular, category 3 or category 4 AMD) intoCNV, and/or the prevention of the development/progression of AMD or CNVin the non- or less effected fellow eye and/or the rate of progressionof the area with GA. In this context, the term “prevention” is used inthe broadest sense to include, complete or partial blocking and slowingdown of the progression of the disease as well as the delay of the unsetof the more serious form of the disease. Patients who are at high riskof developing or progressing into high risk (category 4) AMD or CNVespecially benefit from this aspect of the invention.

Anti-Factor B Antibodies

The invention herein includes the production and use of anti-factor Bantibodies. Exemplary methods for generating antibodies are described inmore detail in the following sections.

Anti-factor B antibodies are selected using a factor B antigen derivedfrom a mammalian species. Preferably the antigen is human factor B.However, factor Bs from other species such as murine factor B can alsobe used as the target antigen. The factor B antigens from variousmammalian species may be isolated from natural sources. In otherembodiments, the antigen is produced recombinantly or made using othersynthetic methods known in the art.

The antibody selected will normally have a sufficiently strong bindingaffinity for the factor B antigen. For example, the antibody may bindhuman factor B with a K_(d) value of no more than about 5 nM, preferablyno more than about 2 nM, and more preferably no more than about 500 pM.Antibody affinities may be determined by a surface plasmon resonancebased assay (such as the BIAcore assay as described in Examples);enzyme-linked immunoabsorbent assay (ELISA); and competition assays(e.g. RIA's), for example.

Also, the antibody may be subject to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody. Examples include the HUVEC inhibitionassay (as described in the Examples below); tumor cell growth inhibitionassays (as described in WO 89/06692, for example); antibody-dependentcellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC)assays (U.S. Pat. No. 5,500,362); and in vitro and in vivo assaysdescribed below for identifying factor B antagonists.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest.

In a preferred embodiment, the anti-factor B antibodies are selectedusing a unique phage display approach. The approach involves generationof synthetic antibody phage libraries based on single frameworktemplate, design of sufficient diversities within variable domains,display of polypeptides having the diversified variable domains,selection of candidate antibodies with high affinity to target factor Bantigen, and isolation of the selected antibodies.

Details of the phage display methods can be found, for example, inWO03/102157 published Dec. 11, 2003.

In one aspect, the antibody libraries can be generated by mutating thesolvent accessible and/or highly diverse positions in at least one CDRof an antibody variable domain. Some or all of the CDRs can be mutatedusing the methods provided herein. In some embodiments, it may bepreferable to generate diverse antibody libraries by mutating positionsin CDRH1, CDRH2 and CDRH3 to form a single library or by mutatingpositions in CDRL3 and CDRH3 to form a single library or by mutatingpositions in CDRL3 and CDRH1, CDRH2 and CDRH3 to form a single library.

A library of antibody variable domains can be generated, for example,having mutations in the solvent accessible and/or highly diversepositions of CDRH1, CDRH2 and CDRH3. Another library can be generatedhaving mutations in CDRL1, CDRL2 and CDRL3. These libraries can also beused in conjunction with each other to generate binders of desiredaffinities. For example, after one or more rounds of selection of heavychain libraries for binding to a target antigen, a light chain librarycan be replaced into the population of heavy chain binders for furtherrounds of selection to increase the affinity of the binders.

Preferably, a library is created by substitution of original amino acidswith variant amino acids in the CDRH3 region of the variable region ofthe heavy chain sequence. The resulting library can contain a pluralityof antibody sequences, wherein the sequence diversity is primarily inthe CDRH3 region of the heavy chain sequence.

In one aspect, the library is created in the context of the humanizedantibody 4D5 sequence, or the sequence of the framework amino acids ofthe humanized antibody 4D5 sequence. Preferably, the library is createdby substitution of at least residues 95-100a of the heavy chain withamino acids encoded by the DVK codon set, wherein the DVK codon set isused to encode a set of variant amino acids for every one of thesepositions. An example of an oligonucleotide set that is useful forcreating these substitutions comprises the sequence (DVK)₇. In someembodiments, a library is created by substitution of residues 95-100awith amino acids encoded by both DVK and NNK codon sets. An example ofan oligonucleotide set that is useful for creating these substitutionscomprises the sequence (DVK)₆ (NNK). In another embodiment, a library iscreated by substitution of at least residues 95-100a with amino acidsencoded by both DVK and NNK codon sets. An example of an oligonucleotideset that is useful for creating these substitutions comprises thesequence (DVK)₅ (NNK). Another example of an oligonucleotide set that isuseful for creating these substitutions comprises the sequence (NNK)₆.Other examples of suitable oligonucleotide sequences can be determinedby one skilled in the art according to the criteria described herein.

In another embodiment, different CDRH3 designs are utilized to isolatehigh affinity binders and to isolate binders for a variety of epitopes.The range of lengths of CDRH3 generated in this library is 11 to 13amino acids, although lengths different from this can also be generated.H3 diversity can be expanded by using NNK, DVK and NVK codon sets, aswell as more limited diversity at N and/or C-terminal.

Diversity can also be generated in CDRH1 and CDRH2. The designs ofCDR-H1 and H2 diversities follow the strategy of targeting to mimicnatural antibodies repertoire as described with modification that focusthe diversity more closely matched to the natural diversity thanprevious design.

For diversity in CDRH3, multiple libraries can be constructed separatelywith different lengths of H3 and then combined to select for binders totarget antigens. The multiple libraries can be pooled and sorted usingsolid support selection and solution sorting methods as describedpreviously and herein below. Multiple sorting strategies may beemployed. For example, one variation involves sorting on target bound toa solid, followed by sorting for a tag that may be present on the fusionpolypeptide (eg. anti-gD tag) and followed by another sort on targetbound to solid. Alternatively, the libraries can be sorted first ontarget bound to a solid surface, the eluted binders are then sortedusing solution phase binding with decreasing concentrations of targetantigen. Utilizing combinations of different sorting methods providesfor minimization of selection of only highly expressed sequences andprovides for selection of a number of different high affinity clones.

High affinity binders for the target factor B antigen can be isolatedfrom the libraries. Limiting diversity in the H1/H2 region decreasesdegeneracy about 10⁴ to 10⁵ fold and allowing more H3 diversity providesfor more high affinity binders. Utilizing libraries with different typesof diversity in CDRH3 (eg. utilizing DVK or NVT) provides for isolationof binders that may bind to different epitopes of a target antigen.

In another embodiment, a library or libraries with diversity in CDRH1,CDRH2 and CDRH3 regions is generated. In this embodiment, diversity inCDRH3 is generated using a variety of lengths of H3 regions and usingprimarily codon sets XYZ and NNK or NNS. Libraries can be formed usingindividual oligonucleotides and pooled or oligonucleotides can be pooledto form a subset of libraries. The libraries of this embodiment can besorted against target bound to solid. Clones isolated from multiplesorts can be screened for specificity and affinity using ELISA assays.For specificity, the clones can be screened against the desired targetantigens as well as other nontarget antigens. Those binders to thetarget NRP1 antigen can then be screened for affinity in solutionbinding competition ELISA assay or spot competition assay. High affinitybinders can be isolated from the library utilizing XYZ codon setsprepared as described above. These binders can be readily produced asantibodies or antigen binding fragments in high yield in cell culture.

In some embodiments, it may be desirable to generate libraries with agreater diversity in lengths of CDRH3 region. For example, it may bedesirable to generate libraries with CDRH3 regions ranging from about 7to 19 amino acids.

High affinity binders isolated from the libraries of these embodimentsare readily produced in bacterial and eukaryotic cell culture in highyield. The vectors can be designed to readily remove sequences such asgD tags, viral coat protein component sequence, and/or to add inconstant region sequences to provide for production of full lengthantibodies or antigen binding fragments in high yield.

A library with mutations in CDRH3 can be combined with a librarycontaining variant versions of other CDRs, for example CDRL1, CDRL2,CDRL3, CDRH1 and/or CDRH2. Thus, for example, in one embodiment, a CDRH3library is combined with a CDRL3 library created in the context of thehumanized 4D5 antibody sequence with variant amino acids at positions28, 29, 30, 31, and/or 32 using predetermined codon sets. In anotherembodiment, a library with mutations to the CDRH3 can be combined with alibrary comprising variant CDRH1 and/or CDRH2 heavy chain variabledomains. In one embodiment, the CDRH1 library is created with thehumanized antibody 4D5 sequence with variant amino acids at positions28, 30, 31, 32 and 33. A CDRH2 library may be created with the sequenceof humanized antibody 4D5 with variant amino acids at positions 50, 52,53, 54, 56 and 58 using the predetermined codon sets.

The anti-factor B antibody generated from phage libraries can be furthermodified to generate antibody mutants with improved physical, chemicaland or biological properties over the parent antibody. Where the assayused is a biological activity assay, the antibody mutant preferably hasa biological activity in the assay of choice which is at least about 10fold better, preferably at least about 20 fold better, more preferablyat least about 50 fold better, and sometimes at least about 100 fold or200 fold better, than the biological activity of the parent antibody inthat assay. For example, an anti-factor B antibody mutant preferably′has a binding affinity for NRP which is at least about 10 fold stronger,preferably at least about 20 fold stronger, more preferably at leastabout 50 fold stronger, and sometimes at least about 100 fold or 200fold stronger, than the binding affinity of the parent anti-factor Bantibodies, such as antibody 20D12.

To generate the antibody mutant, one or more amino acid alterations(e.g. substitutions) are introduced in one or more of the hypervariableregions of the parent antibody. Alternatively, or in addition, one ormore alterations (e.g. substitutions) of framework region residues maybe introduced in the parent antibody where these result in animprovement in the binding affinity of the antibody mutant for theantigen from the second mammalian species. Examples of framework regionresidues to modify include those which non-covalently bind antigendirectly (Amit et al. (1986) Science 233:747-753); interact with/effectthe conformation of a CDR (Chothia et al. (1987) J. Mol. Biol.196:901-917); and/or participate in the V_(L)-V_(H) interface (EP 239400B1). In certain embodiments, modification of one or more of suchframework region residues results in an enhancement of the bindingaffinity of the antibody for the antigen from the second mammalianspecies. For example, from about one to about five framework residuesmay be altered in this embodiment of the invention. Sometimes, this maybe sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant will compriseadditional hypervariable region alteration(s).

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that such randomly produced antibody mutants can bereadily screened.

One useful procedure for generating such antibody mutants is called“alanine scanning mutagenesis” (Cunningham and Wells (1989) Science244:1081-1085). Here, one or more of the hypervariable region residue(s)are replaced by alanine or polyalanine residue(s) to affect theinteraction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The ala-mutants produced this way arescreened for their biological activity as described herein.

Normally one would start with a conservative substitution such as thoseshown below under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity (e.g. bindingaffinity), then more substantial changes, denominated “exemplarysubstitutions” in the following table, or as further described below inreference to amino acid classes, are introduced and the productsscreened. Preferred substitutions are listed in the table below.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Even more substantial modifications in the antibodies biologicalproperties are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr, asn, gln;(3) acidic: asp, glu;(4) basic: his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Nucleic acid molecules encoding amino acid sequence mutants are preparedby a variety of methods known in the art. These methods include, but arenot limited to, oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared mutantor a non-mutant version of the parent antibody. The preferred method formaking mutants is site directed mutagenesis (see, e.g., Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488).

In certain embodiments, the antibody mutant will only have a singlehypervariable region residue substituted. In other embodiments, two ormore of the hypervariable region residues of the parent antibody willhave been substituted, e.g. from about two to about ten hypervariableregion substitutions.

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent antibody, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, and most preferably at least 95%. Identity or similarity withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e sameresidue) or similar (i.e. amino acid residue from the same group basedon common side-chain properties, see above) with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody is determined. As notedabove, this may involve determining the binding affinity and/or otherbiological activities of the antibody. In a preferred embodiment of theinvention, a panel of antibody mutants is prepared and screened forbinding affinity for the antigen such as NRP1 or a fragment thereof. Oneor more of the antibody mutants selected from this initial screen areoptionally subjected to one or more further biological activity assaysto confirm that the antibody mutant(s) with enhanced binding affinityare indeed useful, e.g. for preclinical studies.

The antibody mutant(s) so selected may be subjected to furthermodifications, oftentimes depending on the intended use of the antibody.Such modifications may involve further alteration of the amino acidsequence, fusion to heterologous polypeptide(s) and/or covalentmodifications such as those elaborated below. With respect to amino acidsequence alterations, exemplary modifications are elaborated above. Forexample, any cysteine residue not involved in maintaining the properconformation of the antibody mutant also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant cross linking. Conversely, cysteine bond(s) may beadded to the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment). Another typeof amino acid mutant has an altered glycosylation pattern. This may beachieved by deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

The anti-factor B antibodies of the invention can be producedrecombinantly, using techniques and materials readily obtainable.

For recombinant production of an anti-factor B antibody, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated or synthethized usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to DNAs encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al. (1979) Nature 282:39). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones (1977) Genetics 85:12. The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg (1990) Bio/Technology8:135. Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al. (1991) Bio/Technology 9:968-975.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phos-phate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphor-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phos-phate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al. (1982) Nature 297:598-601 on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus. Alternatively, therous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, a-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv (1982) Nature 297:17-18 on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al. (1977) J. Gen Virol. 36:59); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA77:4216); mouse sertoli cells (TM4, Mather (1980) Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad.Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980)Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal. (1992) Bio/Technology 10:163-167 describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al. (1983) J. Immunol. Meth. 62:1-13). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al. (1986)EMBO J. 5:15671575). The matrix to which the affinity ligand is attachedis most often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

2. Screening Assays and Animal Models for Identifying Factor BAntagonists

Factor B antagonists can be evaluated in a variety of cell-based assaysand animal models of complement-associated diseases or disorders.

Thus, for example, recombinant (transgenic) animal models can beengineered by introducing the coding portion of the genes of interestinto the genome of animals of interest, using standard techniques forproducing transgenic animals. Animals that can serve as a target fortransgenic manipulation include, without limitation, mice, rats,rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g.baboons, chimpanzees and other monkeys. Techniques known in the art tointroduce a transgene into such animals include pronucleicmicroinjection (Hoppe and Wanger, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (e.g., Van der Puttenet al., Proc. Natl. Acad. Sci. USA 82, 6148-615 [1985]); gene targetingin embryonic stem cells (Thompson et al., Cell 56, 313-321 [1989]);electroporation of embryos (Lo, Mol. Cell. Biol. 3, 1803-1814 [1983]);sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73 [1989]).For review, see, for example, U.S. Pat. No. 4,736,866.

For the purpose of the present invention, transgenic animals includethose that carry the transgene only in part of their cells (“mosaicanimals”). The transgene can be integrated either as a single transgene,or in concatamers, e.g., head-to-head or head-to-tail tandems. Selectiveintroduction of a transgene into a particular cell type is also possibleby following, for example, the technique of Lasko et al., Proc. Natl.Acad. Sci. USA 89, 623-636 (1992).

The expression of the transgene in transgenic animals can be monitoredby standard techniques. For example, Southern blot analysis or PCRamplification can be used to verify the integration of the transgene.The level of mRNA expression can then be analyzed using techniques suchas in situ hybridization, Northern blot analysis, PCR, orimmunocytochemistry.

The animals may be further examined for signs of immune diseasepathology, for example by histological examination to determineinfiltration of immune cells into specific tissues. Blocking experimentscan also be performed in which the transgenic animals are treated with acandidate factor B antagonist to determine the extent of effects oncomplement and complement activation, including the classical andalternative pathways, or T cell proliferation. In these experiments,blocking antibodies which bind to the polypeptide of the invention, areadministered to the animal and the biological effect of interest ismonitored.

Alternatively, “knock out” animals can be constructed which have adefective or altered gene encoding factor B, as a result of homologousrecombination between the endogenous gene encoding the factor Bpolypeptide and altered genomic DNA encoding the same polypeptideintroduced into an embryonic cell of the animal. For example, cDNAencoding factor B can be used to clone genomic DNA encoding factor B inaccordance with established techniques. A portion of the genomic DNAencoding factor B can be deleted or replaced with another gene, such asa gene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the factor B polypeptide.

Thus, the biological activity of potential factor B antagonists can befurther studied in murine factor B knock-out mice.

An animal model of age-related macular degeneration (AMD) consists ofmice with a null mutation in Ccl-2 or Ccr-2 gnes. These mice developcardinal features of AMD, including accumulation of lipofuscin in anddrusen beneath the retinal pigmented epithelium (RPE), photoreceptoratrophy and choroidal neovascularization (CNV). These features developbeyond 6 months of age. Candidate factor B antagonists can be tested forthe formation of drusen, photoreceptor atrophy and choroidalneovascularization.

3. Pharmaceutical Compositions

The factor B antagonists of the present invention, including anti-factorB antibodies and other molecules identified by the screening assaysdisclosed above, can be administered for the treatment ofcomplement-associates eye conditions in the form of pharmaceuticalcompositions.

Therapeutic formulations of a factor B antagonist of the invention, areprepared for storage by mixing the active molecule having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. [1980]), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Lipofections or liposomes can also be used to deliver the polypeptide,antibody, or an antibody fragment, into cells. Where antibody fragmentsare used, the smallest fragment which specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable region sequences of an antibody, peptide molecules can bedesigned which retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology (see, e.g. Marasco et al., Proc. Natl. Acad.Sci. USA 90, 7889-7893 [1993]).

The active molecules may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37 C, resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The compounds of the invention for prevention or treatment of an oculardisease or condition are typically administered by ocular, intraocular,and/or intravitreal injection. Other methods administration by also beused, which includes but is not limited to, topical, parenteral,subcutaneous, intraperitoneal, intrapulmonary, intranasal, andintralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration.

Formulations for ocular, intraocular or intravitreal administration canbe prepared by methods and using ingredients known in the art. A mainrequirement for efficient treatment is proper penetration through theeye. Unlike diseases of the front of the eye, where drugs can bedelivered topically, retinal diseases require a more site-specificapproach. Eye drops and ointments rarely penetrate the back of the eye,and the blood-ocular barrier hinders penetration of systemicallyadministered drugs into ocular tissue. Accordingly, usually the methodof choice for drug delivery to treat retinal disease, such as AMD andCNV, is direct intravitreal injection. Intravitrial injections areusually repeated at intervals which depend on the patient's condition,and the properties and half-life of the drug delivered. For intraocular(e.g. intravitreal) penetration, usually molecules of smaller size arepreferred.

The efficacy of the treatment of complement-associated eye conditions,such as AMD or CNV, can be measured by various endpoints commonly usedin evaluating intraocular diseases. For example, vision loss can beassessed. Vision loss can be evaluated by, but not limited to, e.g.,measuring by the mean change in best correction visual acuity (BCVA)from baseline to a desired time point (e.g., where the BCVA is based onEarly Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chartand assessment at a test distance of 4 meters), measuring the proportionof subjects who lose fewer than 15 letters in visual acuity at a desiredtime point compared to baseline, measuring the proportion of subjectswho gain greater than or equal to 15 letters in visual acuity at adesired time point compared to baseline, measuring the proportion ofsubjects with a visual-acuity Snellen equivalent of 20/2000 or worse ata desired time point, measuring the NEI Visual FunctioningQuestionnaire, measuring the size of CNV and amount of leakage of CNV ata desired time point, e.g., by fluorescein angiography, etc. Ocularassessments can be done, e.g., which include, but are not limited to,e.g., performing eye exam, measuring intraocular pressure, assessingvisual acuity, measuring slitlamp pressure, assessing intraocularinflammation, etc.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby expressly incorporated by reference in their entirety.

Example Preparation and Testing of Anti-Factor B Antibody

Commercially available reagents referred to in the example were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the present example, and throughoutthe specification, by ATCC accession numbers is the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209.

Methods

Preparation of Vitreous Fluid and Bruch's Membrane for Protein Analysis

Human AMD and non-AMD cadaver eyes were thawed and the anterior segmentremoved along with the vitreous, retina and RPE. The vitreous wascollected in microtubes, frozen on dry ice and stored at −70 C untilfurther processing. The Bruch's membrane-choroid layer was stripped fromthe posterior half-globe (Crabb, J. W. et al. Proc Natl Acad Sci USA.,99:14682-7 (2002)) and either 4 mm or 6 mm trephined samples wereisolated from the macular and surrounding central region for subsequentanalysis (see FIG. 1). One trephined sample, 4 mm diameter, was used foranalysis of complement factor B protein levels. The sample was sonicatedfor 10 min in Assay Diluent (PBS/0.5% BSA/0.5% Tween-20) and the solubleand insoluble fractions separated by centrifugation for 10 min at 5000rpm. The soluble fraction was used for the ELISA assays.

Generation of Monoclonal Antibodies to Human Factor B

Monoclonal antibodies to human factor B were generated by injecting 2 μgof factor B (Comptech, Taylor, Tex.) in monophosphoryl lipid A/trehalosedicorynomycolate adjuvant (Corixa, Hamilton, Mont.) in the footpads ofBalb/c mice, 11 times. Popliteal lymph nodes from mice were fused withP3X63Ag.U.1 myeloma cells. Hybridoma cells were screened against murinefactor B for binding affinity. Cell lines producing antibodies werecloned by limiting dilution.

Hemolysis Assays

For determining alternative pathway activity, rabbit erythrocytes (Er,Colorado Serum) were washed 3x in GVB and resuspended to 2×10⁹/ml.Inhibitors (50 μl) and 20 μl of Er suspension were mixed 1:1 withGVB/0.1M EGTA/0.1M MgCl₂. Complement activation was initiated by theaddition of C1q-depleted human serum (Quidel; 30 μl diluted 1:3 in GVB).After a 30 minute incubation at room temperature, 200 μl GVB/10 mM EDTAwere added to stop the reaction and samples were centrifuged for 5 minat 500 g. Hemolysis was determined in 200 □l supernatant by measuringabsorbance at 412 nm. Data were expressed as % of hemolysis induced inthe absence of the inhibitor. To determine the effect of CRIg on theclassical pathway of complement, a similar procedure was followed exceptthat Er were replaced with IgM-coated sheep erythrocytes (E-IgM,CompTech) and the assay was performed in factor B deficient human serumin GVB++.

Human Factor B ELISA

Murine anti-human factor Ba mAb (Quidel Corp., Santa Clara, Calif.) wascoated onto plates at 1 μg/mL and biotinylated anti-factor B mAbGNE2FI2.9.3 was used as detection antibody at 800 ng/mL. Intact factor Bprotein (Complement Technology, Inc.) was used as standard with a rangeof 31.25-2,000 pg/mL. SA-HRP was added to the plates at a 1/10,000dilution. The minimum quantifiable concentrations of intact factor B inhuman vitreous fluid and Bruch's membrane lysate samples were 1.56 ng/mL(1/50 minimum dilution) and 312.5 pg/mL (1/10 minimum dilution),respectively.

Molecular Cloning and Reformatting of Monoclonal Anti-Factor B Antibody1F7

Total RNA was extracted from hybridoma cells producing the mouseanti-human factor B monoclonal 1F7, using RNeasy Mini Kit (Qiagen,Germany). The variable light (VL) and variable heavy (VH) domains wereamplified using RT-PCR with the following degenerate primers:

Light chain (LC) forward: (SEQ ID NO: 4)5′GGAGTACATTCAGATATCGTGCTGACCCAATCTCCAGCTTCTTTGGC T3′Light chain reverse: (SEQ ID NO: 5)5′GGTGCAGCCACGGTCCGTTTGATTTCCAGCTTGGTGCCTCCACC3′Heavy chain (HC) forward: (SEQ ID NO: 6)5′GGAGTACATTCACAGATCCAGCTGGTGCAGTCTGGACC3′ Heavy chain reverse:(SEQ ID NO: 7) 5′GACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGASTGWGGTT CC3′

The forward primers were specific for the N-terminal amino acid sequenceof the VL and VH region. Respectively, the LC and HC reverse primerswere designed to anneal to a region in the constant light (CL) andconstant heavy domain 1 (CH1), which is highly conserved across species.

Amplified VL was cloned into a pRK mammalian cell expression vector(Shields et al., J Biol Chem 276: 6591-6604 (2001)). containing thehuman kappa constant domain. Amplified VH was inserted to a pRKmammalian cell expression vector encoding the full-length human IgG1constant domain. Thus, 1F7 was reformatted to a mouse-human IgG1chimera.

The plasmids were grown in E. coli and expressed in Chinese hamsterovary (CHO) cells.

What is claimed is:
 1. An anti-factor B antibody binding essentially tothe same epitope as anti-factor B antibody 1F7.
 2. An anti-factor Bantibody comprising the light and/or heavy chain hypervariable regionsequences of anti-factor B antibody 1F7 (SEQ ID NOs: 1 and 2,respectively).
 3. An anti-factor B antibody comprising the light and/orheavy chain variable region sequences of anti-factor B antibody 1F7 (SEQID NOs: 1 and 2, respectively).
 4. The anti-factor B antibody of claim 3which is antibody 1F7 comprising a light chain sequence of SEQ ID NO: 1and the heavy chain sequence of SEQ ID NO:
 2. 5. The anti-factor Bantibody of any one of claims 1-4, which is a monoclonal antibody. 6.The anti-factor B antibody of claim 5, which is an antibody fragment. 7.The anti-factor B antibody of claim 6 wherein the antibody fragment isselected from the group consisting of Fab, Fab′, F(ab′)₂, saFv, (scFv)₂,dAb, complementarity determining region (CDR) fragments, linearantibodies, single-chain antibody molecules, minibodies, diabodies, andmultispecific antibodies formed from antibody fragments.
 8. Theanti-factor B antibody of claim 5, which is chimeric, humanized orhuman.
 9. The anti-factor B antibody fragment of claim 6, which ischimeric, humanized or human.
 10. A method for the prevention ortreatment of a complement-associated eye condition comprisingadministering to a subject in need an effective amount of a factor Bantagonist.
 11. The method of claim 10 wherein said said subject is amammal.
 12. The method of claim 11 wherein said subject is a human. 13.The method of claim 12 wherein said factor B antagonist is selected fromthe group consisting of anti-factor B antibodies and fragments thereof,binding polypeptides, peptides, and non-peptide small molecules.
 14. Themethod of claim 13 wherein said factor B antagonist is an antibody or anantibody fragment.
 15. The method of claim 14 wherein said antibody orantibody fragment binds essentially to the same epitope as anti-factor Bantibody 1F7.
 16. The method of claim 14 wherein said antibody orantibody fragment comprises the light and/or heavy chain hypervariableregion sequences of anti-factor B antibody 1F7 (SEQ ID NOs: 1 and 2,respectively).
 17. The method of claim 14 wherein said antibody orantibody fragment comprises the light and/or heavy chain variable regionsequence of anti-factor antibody 1F7 (SEQ ID NOs: 1 and 2,respectively).
 18. The method of claim 14 which is antibody 1F7comprising a light chain sequence of SEQ ID NO: 1 and the heavy chainsequence of SEQ ID NO:
 2. 19. The method of claim 14 wherein saidantibody or antibody fragment binds to the active site of factor B. 20.The method of claim 14 wherein said antibody or antibody fragment bindsto an epitope including active site residues of factor B.
 21. The methodof claim 14 wherein said antibody fragment is selected from the groupconsisting of Fab, Fab′, F(ab′)₂, scFv, (scFv)₂, dAb, complementaritydetermining region (CDR) fragments, linear antibodies, single-chainantibody molecules, minibodies, diabodies, and multispecific antibodiesformed from antibody fragments.
 22. The method of claim 21 wherein saidantibody fragment is a Fab, Fab′, F(ab′)₂, scFv, or (scFv)₂ fragment.23. The method of claim 10 wherein said complement-associated eyecondition is selected from the group consisting of age-relared maculardegeneration (AMD), choroidal neovascularization (CNV), uveitis,diabetic and other ischemia-related retinopathies, diabetic macularedema, pathological myopia, von Hippel-Lindau disease, histoplasmosis ofthe eye, Central Retinal Vein Occlusion (CRVO), cornealneovascularization, and retinal neovascularization.
 24. The method ofclaim 23 wherein said AMD is dry AMD.
 25. The method of claim 23 whereinsaid AMD is wet AMD.
 26. A kit comprising a factor B antagonist andinstructions for administering said antagonist to treat acomplement-associated eye condition.
 27. The kit of claim 26 whereinsaid complement-associated eye condition is selected from the groupconsisting of age-relared macular degeneration (AMD), choroidalneovascularization (CNV), uveitis, diabetic and other ischemia-relatedretinopathies, diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, Central Retinal VeinOcclusion (CRVO), conical neovascularization, and retinalneovascularization.
 28. The kit of claim 27 wherein saidcomplement-associated eye condition is AMD or CNV
 29. The use of afactor B antagonist in the preparation of a medicament for the treatmentof a complement-associated eye condition.
 30. A factor B antagonist foruse in the treatment of a complement-associated eye condition.